Method for concentrating beta-glucan film

ABSTRACT

An entirely aqueous method for concentrating beta-glucan from a beta-glucan source, such as milled cereal bran, grain or distiller&#39;s dried grain. The method comprises providing an alkaline aqueous extract of a beta-glucan source; acidifying or neutralizing the extract and heating the extract to between about 60° C. and 100° C.; cooling the extract, whereby a flocculate is formed; acidifying the cooled extract if the extract was neutralized; and removing the flocculate from the aqueous solution to form an intermediate solution. The intermediate solution may be subjected to ultrafiltration for further purification of beta-glucan, or may be evaporated, resulting in formation of a solid film enriched in beta-glucan. Beta-glucan has cholesterol-lowering properties and is a topical immunostimulant.

RELATED APPLICATIONS

[0001] This application is a continuation of co-pending U.S. patentapplication Ser. No. 09/921,846, filed on August 2, 2001, which is adivisional of U.S. patent application Ser. No. 09/252,356, now U.S. Pat.No. 6,323,338, filed on Feb. 17, 1999.

FIELD OF THE INVENTION

[0002] The present invention relates to a method for isolating andconcentrating mixed-linkage beta-glucans from a beta-glucan source, suchas cereal bran or grain, particularly oats and barley. The disclosedprocess is entirely aqueous and results in efficient production ofbeta-glucan concentrates. This is a divisional application of U.S.application Ser. No. 09/252,356 filed on Feb. 17, 1999.

BACKGROUND OF THE INVENTION

[0003] Mixed-linkage (1

3), (1

4) beta-D-glucans, referred to herein as beta-glucan, are thepredominant cell wall components of grain endosperm, particularly oatsand barley, and are a well-established antihypercholesterolemic agent.In the case of beta-glucan found naturally in oats, this effect has beenacknowledged by the U.S. Food and Drug Administration (FDA). Beta-glucanalso has immunostimulatory properties when applied topically to theskin. The biochemical mechanisms by which beta-glucan exerts itstherapeutic effects are largely unknown.

[0004] Cereal grain seeds generally contain a small amount ofbeta-glucan, with oats and barley being recognized as the richestsources of this material. The naked oat seed, known in the art as a“groat”, typically contains from 2-4% by weight beta-glucan, dependingupon oat variety and other factors such as growing conditions. Barleyseeds may typically contain twice as much beta-glucan as groats.Beta-glucan is generally found in higher concentrations in the outermostlayers of the seed (i.e., the “bran”). Thus, oat bran is defined ascontaining a minimum of 5.5% by weight beta-glucan, and typicallycontains up to 6% or 7% by weight beta-glucan.

[0005] In order to receive an efficacious amount of beta-glucan forreduction of low density lipoprotein (LDL) and total serum cholesterol,the FDA recommends total beta-glucan ingestion of at least 3 gramsdaily. However, it is difficult and inconvenient for the averageindividual to obtain this recommended amount because of the inherentlylow beta-glucan content in products such as oatmeal, oat bran muffins orcooked barley. For example, in the case of oatmeal, which is simplyrolled whole oats, one would have to consume up to 150 grams (dry basis)each day, an amount which most individuals would find extremelyburdensome. Accordingly, there is a compelling need to provide a moreconcentrated form of beta-glucan so that consumers can convenientlyingest therapeutic amounts of this material.

[0006] Previous processes for concentrating beta-glucan from cerealssuch as oats or barley have proven impractical for commercialmanufacturing processes because of high cost and/or low yields. Theprohibitive cost associated with processes disclosed in the literatureis almost always a consequence of reliance upon a precipitation step inwhich beta-glucan is removed from aqueous solution by an organicsolvent, especially alcohols such as ethanol or isopropanol, the use ofwhich entails high in-process losses and difficult reclamation. This isevidenced in the marketplace wherein beta-glucan concentrates are onlysold as cosmetic ingredients, with prices typically greater than $100per pound. There is presently no concentrated form of beta-glucan pricedso as to be affordable for use as a dietary supplement or food additive.The most concentrated form of commercially available beta-glucan fornutritional purposes contains just 15% beta-glucan (Nurture® 1500,supplied by Nurture, Inc., Missoula, Mont.).

[0007] U.S. Pat. No. 5,518,710 to Bhatty discloses alkaline extractionof barley and oat bran, addition of an amylolytic agent to degradestarches, followed by precipitation of beta-glucan with a polar alcohol.Beer et al. (Cereal Chemistry 73:58-62, 1996) disclose a process forisolating beta-glucan in which oat bran concentrates are extracted inaqueous solution at alkaline pH, followed by dialysis, ultrafiltrationor alcoholic precipitation. The material resulting from these processeshad a beta-glucan content of about 60-65%. Westerlund et al.(Carbohydrate Polymers, 20:115-123, 1993) disclose a procedure forisolation of beta-glucan involving lipid extraction, enzymatic removalof starch and protein, and subsequent ethanol precipitation. EP 0 377530 A2 discloses a process for the preparation of a beta-glucan-enrichedgrain for use as a food or food additive, in which oats are slurried incold water, followed by rapid homogenization and screening of theslurry. U.S. Pat. No. 5,013,561 to Goering et al. discloses a processfor recovery of various products, including beta-glucan, from barley. Inthis process, barley is milled, mixed with water, heated and centrifugedto remove insoluble material. The supernatant is then heated, andcentrifuged to remove insoluble material, and the supernatant issubjected to ultrafiltration to remove soluble sugars and to concentratebeta-glucan solids.

[0008] There is a need for an entirely aqueous process for concentratingbeta-glucan which is efficient, economical and produces highly palatablebeta-glucan concentrates for use in foods and pharmaceuticalformulations. The present invention addresses this need.

SUMMARY OF THE INVENTION

[0009] One embodiment of the present invention is a method forconcentrating beta-glucan from a beta-glucan source, comprising thesteps of: (a) providing an alkaline aqueous extract of the beta-glucansource; (b) acidifying or neutralizing the extract and heating theextract to between about 60° C. and 100° C.; (c) cooling the extract,whereby a flocculate is formed; (d) acidifying the cooled extract if theextract was neutralized in step (b); and (e) removing the flocculatefrom the extract to form an intermediate solution. Preferably, thebeta-glucan source is cereal grain, cereal bran, milled cereal grain,milled cereal bran or distiller's dried grain (DDG). Advantageously, thecereal is oats or barley.

[0010] In one aspect of this preferred embodiment, the aqueous alkalineextract has a pH of between about 7.5 and 12. Preferably, the aqueousalkaline extract has a pH of about 10. In another aspect of thispreferred embodiment, the cereal grain is extracted at a temperature ofbetween about 25° C. and 80° C. Advantageously, the extract of step (c)is cooled to between about 25 and 45° C. In one aspect of this preferredembodiment, the flocculate is removed by centrifugation. In addition, anamylolytic enzyme may be added during step (b). The method may furthercomprise subjecting the intermediate solution to ultrafiltration.Preferably, the intermediate solution is heated prior toultrafiltration. In one aspect of this preferred embodiment, theintermediate solution is subjected to diafiltration prior to theultrafiltration step. In another aspect of this preferred embodiment,the intermediate solution is treated with activated carbon or ionexchange media. The method may further comprise the step of drying theintermediate solution. Preferably, the drying step is performed in adouble drum dryer or spray dryer. The method may further comprise thestep of heating the intermediate solution to allow evaporationtherefrom, whereby a solid film or skin, enriched in beta-glucan, isformed on the surface of the solution. In addition, the method mayfurther comprise the step of removing the beta-glucan film, resulting information of a second beta-glucan film. Preferably, the beta-glucan filmremoving step is performed one or more times. The method may furthercomprise drying the beta-glucan film. Another aspect of this preferredembodiment further comprises the step of subjecting the intermediatesolution to ultrafiltration after removal of the beta-glucan film.Preferably, the ultrafiltration step removes salts and othercontaminants from the intermediate solution.

[0011] Another embodiment of the present invention is substantiallyconcentrated beta-glucan produced by the methods described above.

[0012] The present invention also provides an isolated beta-glucanenriched solid film or concentrate produced by the methods describedabove.

[0013] The present invention also provides a composition comprisingbeta-glucan produced by any of the methods described above, incombination with a food product.

[0014] The present invention also provides a pharmaceutical compositioncomprising beta-glucan produced by any of the methods described above,and a pharmaceutically acceptable carrier.

[0015] In addition, the present invention provides a cosmeticcomposition comprising beta-glucan produced by any of the methodsdescribed above.

[0016] Another embodiment of the present invention is an isolatedbeta-glucan-enriched film formed by evaporating a beta-glucan-containingsolution until surface film formation occurs. The beta-glucan-enrichedfilm may be provided in combination with a food product. Thebeta-glucan-enriched film may also be provided as a pharmaceuticalcomposition in combination with a pharmaceutically acceptable carrier.The beta-glucan may also be provided as a cosmetic composition.

[0017] The present invention also provides a method for preparingconcentrated beta-glucan by evaporating water from an aqueousbeta-glucan-containing solution, and allowing a solid film ofconcentrated beta-glucan to form on the surface of the solution.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 is a diagram summarizing the production ofbeta-glucan-enriched intermediate solution. Oat bran is milled, slurriedand passed over a screen to remove starch. The material unable to passthrough the screen is extracted under alkaline conditions andcentrifuged. The supernatant is acidified, heated, cooled andcentrifuged to remove proteins. The resulting supernatant is called theintermediate solution. It should be noted that oat bran is only oneexemplary source of beta-glucan.

[0019]FIG. 2 is a diagram showing three processing pathways for theintermediate solution produced as shown in FIG. 1. The intermediatesolution is dried and milled to form a 35%-55% beta-glucan concentrate(left pathway), evaporated to produce a skin enriched in beta-glucan,which is dried and milled to form a 50-95% beta-glucan concentrate(center pathway) or subjected to ultrafiltration, dried and milled toproduce a 50-95% beta-glucan concentrate (right pathway) it should benoted that the dryng step in each pathway is optional.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0020] The present invention provides an improved, totally aqueousprocess for concentration of beta-glucan from any beta-glucan-containingsource including, but not limited to, cereal grains and brans,particularly oats and barley. Typically, the cereal grains and brans aremilled prior to concentration of beta-glucan. The term “cereal” includes(but is not limited to) any of the various cultivars of oat, barley,wheat, rye, corn, rice, sorghum, millet and amaranth. The term“beta-glucan” is defined as a glucan with a mixed β(1

3), (1

4) linked glucopyranosyl backbone.

[0021] The present method can also be used to produce beta-glucanconcentrates from distiller's dried grain (DDG), also known as spentbrewer's grain, which is the spent, dried grains recovered after alcoholfermentation of cereals, particularly wheat, corn and barley. DDG islargely a waste material with few commercial uses.

[0022] The process described herein for concentrating beta-glucan iscompletely aqueous, and does not require any organic or toxic solvents,thus enabling production of concentrated beta-glucan at a manufacturingcost sufficiently low to enable sale of the product for nutritionalapplications. The resulting beta-glucan retains a high molecular weight,between about 400,000 and one million daltons, which is believed to beessential for its cholesterol lowering ability.

[0023] Various parameters of the present beta-glucan concentrationmethod can also be adjusted to vary the final beta-glucan concentrationfrom about 30% weight to more than 90% weight. In a preferredembodiment, the concentration of beta-glucan produced by the presentmethod is between about 30% weight and about 100% weight. In a morepreferred embodiment, the concentration is between about 50% and about95%. In a most preferred embodiment, the concentration is between about60% and about 90%. The resulting beta-glucan concentrate is entirelywater soluble, thereby facilitating its use in a variety of dosageforms.

[0024] The present method is scalable and, accordingly, processequipment can be sized sufficiently large so as to maximize economies ofscale. The method also generates several co-product streams rich instarch, proteins and lipids which are valuable in nutritional andpersonal care applications. The resulting beta-glucan is also useful asa cosmetic ingredient.

[0025] The beta-glucan concentration method of the present invention isillustrated in the diagrams shown in FIGS. 1 and 2. Referring to FIG. 1,oat bran, or any other desired cereal grain bran, is ground or milled,i.e., size reduced, using hammer mill 2 to form a bran flour. The oatbran may also be milled in any other conventional manner known in thegrain milling art (e.g., pin milling). Optionally, the whole grain maybe inactivated prior to or after milling to minimize glucanase activity,such as by heating to about 100° C. Whole grains, cereal flours, bransand commercially available breakfast cereals can also be used asstarting materials in the process of the present invention. Cerealflours and brans are commercially available from a number of sources orcan be produced from a desired cereal using standard milling techniquessuch as described in Bhatty, Cereal Chem. 63:31-35, 1986; Wood et al.,Cereal Chem. 66:97-103, 1989; and Wood et al., Cereal Chem. 68:31-39,1991, and are well known to those of skill in the art.

[0026] The milled bran is then slurried with an aqueous solution,preferably cold water, in a slurry tank 4, or other suitable vessel, andpumped over a screen or filter, such as a USS 100 vibratory screen(149:m opening) 6, to remove starch, although vibratory screens with USSmesh sizes between about 35 and 400 are suitable for use in the presentinvention. The fine starch granules (“unders”), typically less than 20microns in size, pass through the screen, forming a starchy coproduct,while the milled bran (“overs”) is much more coarse and does not passthrough the screen. Other wet or dry methods for removing starch and/orother undesirable components are also contemplated, including airclassification, differential density or electrostatic techniques. Thematerial which does not pass through the screen is extracted underalkaline conditions in a heated extractor 8, or other suitable vessel inorder to dissolve beta-glucan. The alkaline extraction is preferablyperformed at a pH between about 7.5 and 12, more preferably betweenabout 9 and 11, and most preferably at a pH of about 10. Although theuse of sodium carbonate for beta-glucan extraction is described herein,any organic or inorganic base is suitable for use in the presentinvention, including, but not limited to, sodium hydroxide, potassiumhydroxide, sodium bicarbonate, ammonia and the like. The alkalineextraction is generally performed at a temperature of between about 40°C. and 80° C., more preferably between about 50° C. and 70° C. and mostpreferably between about 55° C. and 65° C. for between about 0.5 hourand 3 hours, preferably about 1 or 2 hours.

[0027] Undissolved solids, including starch, insoluble fiber and otherinsoluble constituents of the spent bran, are removed by centrifuge 10,although the use of other conventional separation techniques includingdialysis, filtration and passage through a mesh or cloth are alsocontemplated. This results in formation of a supernatant and a brannycoproduct. Optionally, this branny coproduct may be recycled to theheated extractor 8 for one or more extractions, followed by removal ofundissolved solids. The supernatant (or combined supernatants) is theneither acidified to a pH of between about 2 and 6, preferably about 4(to induce isoelectric precipitation of proteins), with an organic orinorganic acid (e.g., hydrochloric, acetic, phosphoric, citric and thelike), or neutralized with an organic or inorganic acid to a pH ofbetween about 6 and 8, preferably about 7, and heated to or near theboiling point (between about 60° C. and 100° C., preferably betweenabout 80° C. and 90° C.) for between about 15 minutes and 1 hour,preferably 30 minutes, in a heating tank 12 or other suitable vessel inorder to eliminate bacteria and neutralize any residual glucanaseactivity. Heating of the supernatant to or near the boiling pointpromotes the subsequent precipitation of various solids, includingproteins, in the cooling step described below. An amylolytic enzyme suchas α-amylase (e.g., Termamyl) may optionally be added during this stepin order to eliminate, in whole or in part, contaminating starches. Thesolution is then cooled to between about 25° C. and 45° C., preferablyto about 30° C., resulting in formation of a heavy flocculate mostlikely comprising primarily protein and starch (proteinaceouscoproduct). It should be noted that the flocculate generally forms uponcommencement of the cooling process, except in the case where thesolution has been neutralized rather than acidified as described above,in which case the flocculate may form either prior to or afteracidification in the cooling step. The cooling step is typicallyperformed in a heat exchanger 14, although other methods includingplacing the solution in a water bath or allowing the solution to cool byitself are also within the scope of the invention. If the supernatantwas neutralized prior to heating to near the boiling point, theresulting material is acidified to a pH of between about 2 and 6,preferably about 4, after the cooling step.

[0028] The flocculate is then removed, preferably by centrifuge 10,although the other solid removal methods listed above can also be used.The resulting solution may be optionally refined by conventionaltreatment with activated carbon or ion exchange media.

[0029] The resulting solution, referred to as the “intermediate”solution, contains between about 0.1% and 3.0%, preferably between about0.5% and 1.0% weight dissolved solids, comprising beta-glucan, starch,protein and salts resulting from prior acid/base additions. Theintermediate solution may be dried using conventional drying equipmentsuch as a vacuum evaporator 16 and double-drum dryer 18, or a spraydryer (FIG. 2). The beta-glucan content of the dried product istypically between about 35 and 55% weight, and may range from about25-55% weight. The dried product is then milled using mill 2. Theproduct also contains a significant amount of salt, and is thus usefulfor dietary products in which it replaces added salt. However, in otherpreferred embodiments, the majority of salt and other low molecularweight contaminants are removed, thereby increasing the beta-glucanconcentration and improving palatability and versatility.

[0030] As described in Example 6 below, the step in which thesupernatant is heated to near the boiling point, after eitheracidification or neutralization, unexpectedly results in significantlymore beta-glucan in the supernatant after cooling and removal of theresulting flocculate than does the conventional step disclosed in theliterature, in which the supernatant is cooled to ambient temperature,followed by acidification to pH 4 and subsequent removal of precipitatedsolids by centrifugation. This conventional step typically precedesbeta-glucan precipitation in alcohol in prior art processes.

[0031] In a preferred embodiment of the present invention, theintermediate solution is placed in ultrafiltration unit 22 for furtherconcentration of beta-glucan using a membrane with an appropriate poresize to retain beta-glucan (FIG. 2). Membranes suitable for use in theultrafiltration step have pore sizes ranging from between about 200angstroms and 5:m, preferably about 0.2:m. This method is used fordewatering purposes because it can be more cost-effective thantraditional thermal drying techniques. In the present method,ultrafiltration using well known crossflow ceramic filters, polymerfilters, and the like is used for conventional dewatering and forremoval of low molecular weight contaminants, such as sodium chloride,from the intermediate solution. Optionally, the solution may be heatedin a container including, but not limited to, heating tank 20 to betweenabout 80° C. and 95° C., preferably about 90° C., and/or fresh water maybe added (i.e., diafiltration) prior to ultrafiltration in order toincrease filtration efficiency (i.e., reduced viscosity and fouling).The ultrafiltered solution (retentate), when dried by conventional means(i.e., double drum dryer 18), yields a concentrate typically containingbetween about 60% and 70% weight beta-glucan; however, higherbeta-glucan concentrations, preferably 75%, 80%, 85%, 90% or 95%, can beobtained by repeated rounds of ultrafiltration or by routineoptimization of process conditions. Overall beta-glucan recovery, on thebasis of the beta-glucan initially contained in the starting rawmaterial, is as high as about 70%. The dried material is then milledusing mill 2.

[0032] Unexpectedly, when the intermediate solution was heated in one ormore open vessel(s) such as tray evaporator 24, or any other suitableopen vessel such as a bowl, vat or beaker, a thin, solid film, or“skin”, spontaneously formed on the surface of the liquid, and generallycovered the entire surface (FIG. 2). This surface skin, which was clearand translucent in appearance, comprised predominantly beta-glucan andseparated out from a very dilute beta-glucan solution. It is notessential that the vessel be open during skin formation. Any atmosphereabove the vessel that permits sufficient skin formation can be used. Inaddition, the evaporation step may be performed under continuouscountercurrent flow utilizing fresh water or other aqueous solutions.

[0033] Without wishing to be bound by any particular theory, it isbelieved that the formation of the beta-glucan skin is due to eitherhighly localized saturation or supersaturation of beta-glucan insolution resulting from evaporation, leading to the rapidcrystallization or precipitation of beta-glucan. It appears that bothheating and evaporation are required for skin formation. The evaporationmay provide a cooling effect, thereby promoting formation of the film atthe surface, or may simply provide a removal mechanism for newly unboundwater. After removal of the beta-glucan skin from the surface of thesolution, and upon further heating and evaporation of the solution, asecond beta-glucan skin is formed and removed. This process can berepeated one or more times to isolate additional beta-glucan skin. Thismultiple skin formation is described in Examples 2, 5 and 8.

[0034] The term “self-assembly” has been adopted to describe mechanismby which beta-glucan skin formation may occur. It is believed that ahomogeneous beta-glucan solution of virtually any concentration can bemade inhomogeneous by appropriate heating and attendant evaporation.Thermal convection currents are believed to convey the beta-glucanmolecules to the liquid surface. There appears to be a driving force forthese molecules to decrease the entropy of the system by congregating atthe surface. This driving force may arise from surface tension effectsand/or initiation of hydrogen bonding sufficient to herd the moleculestogether.

[0035] Formation of a viscous layer at the surface just prior to skinformation was also observed, which is in accord with the mechanismdescribed above. This layer appears to be one or two millimeters thick.This layer has been harvested and found to contain 34.0% w beta-glucan(0.97% w solids), whereas the homogeneous parent material contained only27.3% w beta-glucan (0.70% w solids). It is not unlikely that there is apronounced concentration gradient within the viscous layer whereinhighly concentrated beta-glucan is found in the uppermost (i.e., closestto the surface) molecular layers.

[0036] Thermal energy being applied to the solution may promote thedisplacement of bound water molecules and allow closer mutual approachof beta-glucan molecules. The proximity of an air-water interfacefacilitates removal of the newly unbound water. At some critical point,the beta-glucan molecules may approach closely enough to allow hydrogenbonding, and the beta-glucan then is believed to self-assemble into itspreferred, planar conformation. This conformation is discussed in anx-ray diffraction study by Tvaroska et al. (Can. J Chem. 61:1608-1616,1983). Effectively, the beta-glucan becomes insoluble at this point andforms a solid phase having a density less than water, causing it tofloat. It should be noted that self-assembly of the beta-glucanmolecules into planar form may actually be initiated well below theliquid surface and may be thermally driven; the partially assembledmolecules may be less dense than water, further facilitating their riseto the surface. The solid phase can only be re-dissolved by applicationof sufficient energy for re-insertion (and re-binding) of water betweenbeta-glucan molecules. It is also believed that relatively quiescentconditions are necessary for the solid planar phase to form, or, atleast, to be readily visible. In fact, this aspect of the invention hasprobably deterred discovery by others, given the vigorous agitationnormally associated with processes for beta-glucan recovery.

[0037] This beta-glucan skin may be rinsed with water one or more times(26), and dried in product dryer 28 to form a beta-glucan concentrate.If desired, the product may also be milled in mill 2. Thus, filmformation provides another commercially viable basis for furtherprocessing of the intermediate solution. Although the beta-glucansurface skin was first observed in an unstirred surge tank containingintermediate solution which had been ultrafiltered, it was also foundthat the unfiltered intermediate solution would form a surface skin. Thesurface skin has also been induced using extract supernatant.

[0038] In addition, skin formation was also observed after slurrying oatbran in water for one hour and heating the resulting suspension in anopen container to allow evaporation. This skin contained 17.6%beta-glucan. Although the beta-glucan concentration was diluted by thepresence of random floating (brownish) masses of branny material, thisrepresents a significant enrichment. In addition, the viscous thin layerprior to film formation was found to contain 9.6% beta-glucan. Thus, anybeta-glucan-containing solution can be used for skin formation ifallowed to evaporate.

[0039] The film can be readily harvested by various skimming orfiltering techniques. Significantly, with continued heating, the film israpidly regenerated on the surface. In this way, a significant quantityof beta-glucan in a substantially concentrated form can be isolated bysuccessive harvests of film. It is advantageous to employ an evaporationvessel with a high surface area to volume ratio for this purpose.Overall beta-glucan recovery of at least 50% can be achieved using thefilm formation method. Beta-glucan concentrations of between about 60and 85% weight in the dried film are routinely obtained. Furtherconcentration increases, up to about 95% weight, may be obtained byminimal water washing of the film subsequent to harvest in order toremove excess dissolved contaminants. One particular advantage of theharvested film is that most of the salts and other dissolvedcontaminants remain in the heated solution. Moreover, the harvested filmcan be readily dried with conventional equipment and with minimal energybecause the film is already substantially dewatered.

[0040] Optionally, for purposes of optimizing beta-glucan recovery,fresh water may be added to the beta-glucan-depleted, increasingly brinysolution. This can be done in, for example, a standard countercurrentflow arrangement, involving one or more evaporation vessels, so as toachieve steady-state film regeneration and recovery, which isparticularly useful for continuous flow production. The remainingsolution can be further refined to recover and concentrate additionalbeta-glucan by sending it through an ultrafiltration unit as describedabove, thereby constituting a hybrid process.

[0041] The concentration techniques described above may be configured torun on either a batchwise basis or a continuous flow basis. Continuousflow is preferred because of the economies inherent in a continuousoperating mode. These techniques can also be applied to any othersources of beta-glucan with minor modifications.

[0042] The beta-glucan produced by the methods of the present inventionis useful in lowering serum cholesterol levels, and can be incorporatedinto pharmaceutical formulations, cosmetic formulations, or combinedwith food products. Because the beta-glucan produced by the presentmethod is highly palatable, it can be added to a variety of foodsincluding dairy products, dips, spreads, powdered drink mixes,margarine, packaged mixes, confections, health bars, soups, dressings,and the like. It can also be incorporated into pharmaceuticallyacceptable excipients or diluents, and into pharmaceutical formulationsincluding tablets, capsules, elixirs, syrups, lozenges and the like.Beta-glucan-containing cosmetic formulations include skin lotions,soaps, shampoos, hair conditioners, skin creams and the like.

[0043] For oral administration, the beta-glucan may be incorporated intoa tablet, aqueous or oil suspension, dispersible powder or granule,microbead, emulsion, hard or soft capsule, syrup or elixir. Compositionsmay be prepared according to any method known in the art for themanufacture of pharmaceutically acceptable compositions, such as thosedescribed in Remington's Pharmaceutical Sciences, 15th edition, MackPublishing Co., Easton, Pa., 1985. These compositions may contain one ormore of the following agents: sweeteners, flavoring agents, coloringagents and preservatives. Tablets containing the active ingredients inadmixture with non-toxic pharmaceutically acceptable excipients suitablefor tablet manufacture are acceptable. “Pharmaceutically acceptable”means that the agent should be acceptable in the sense of beingcompatible with the other ingredients of the formulation (as well asnon-injurious to the individual). Such excipients include inert diluentssuch as calcium carbonate, sodium carbonate, lactose, calcium phosphateor sodium phosphate; granulating and disintegrating agents, such as cornstarch and alginic acid; binding agents such as starch, gelatin oracacia; and lubricating agents such as magnesium stearate, stearic acidor talc. Tablets may be uncoated or may be coated with known techniquesto delay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period of time. Forexample, a time delay material such as glyceryl monostearate or glycerylstearate alone or with a wax may be employed.

[0044] The beta-glucan prepared by the methods of the present inventionmay also be incorporated into topical formulations for use as animmunostimulant. Such topical formulations include creams, ointments,gels, salves, pastes, lotions, suspensions, emulsions and dispersions.

[0045] When wet or dissolved, the beta-glucan or beta-glucan-containingsolutions of the present invention feel oily and have a mouthfeelsimilar to that of fatty foods. It is therefore contemplated that thesecompositions can be used as fat substitutes.

[0046] The following examples describing the beta-glucan concentrationmethod of the present invention are meant to be construed asillustrative rather than limiting.

EXAMPLE 1 Preparation of Beta-Glucan-Containing Intermediate Solution

[0047] Commercial oat bran (Con Agra SCM-350) was fed into an Ajax 17Dhammer mill fitted with a 2.5/64-inch screen. The resultant flour (50lb) was added at a rate of 1.5 lb/min to a 20-gallon stainless steeltank where it was slurried with 15° C. water to approximately 10% wsolids concentration. The flour/water slurry was pumped to a vibratoryscreen (Rotex Model 3431, 40×84″) fitted with an overhead cold waterspray bar and a USS 100 screen (149:m openings). The oversized materialfrom the screen was gravity-fed to a 230-gallon stainless steel,jacketed extractor fitted with a propeller agitator. Sufficient waterwas added to result in 100-gallons of mixture. Temperature wasmaintained at 60° C. and pH was maintained at 10 by addition of 20% wsodium carbonate solution. The mixture was extracted under theseconditions for two hours. Extraction mass exiting the vessel was pumpedto a decanter centrifuge (Bird HP200) in order to remove solids.Supernatant fluid from the centrifuge was discharged to another230-gallon stainless steel jackCted vessel equipped with propelleragitation. Temperature in this vessel was maintained at 80° C. and pHwas maintained at 4 by addition of 20% HCl. The supernatant was heldunder these conditions for 15 minutes. Material exiting the vessel wasconveyed through the shell side of a stainless steel shell-and-tube heatexchanger and was thereby cooled to less than 30° C. The cooled fluidwas fed to a disk centrifuge (Westfalia SAMN 5036) in order to removeprecipitated solids. The fluid exiting from the centrifuge was called“intermediate solution” and contained 0.72% w solids. These solidscontained 42.9% w beta-glucan as determined using the modified McClearymethod which is a standard assay for mixed linkage (1

3), (1

4) beta-glucan. This is a dual enzyme technique which reducesmacromolecular beta-glucan to glucose. The glucose is reacted to form achromophore which is subsequently quantitated by spectrophotometry. Itis a codified method of the American Association of Cereal Chemists(AACC Method 32-33). This method was used to determine beta-glucancontent in all of the examples provided below.

EXAMPLE 2 Formation and Harvesting of Beta-Glucan “Skin”

[0048] Intermediate solution prepared in accordance with Example 1 wasplaced in a steam-heated stainless steel tray having a relatively largesurface area to volume ratio (23″ wide×48″ long×7.25″ deep). Theintermediate solution was maintained at a temperature of 87° C. The traywas initially filled with solution to a depth of 3 inches. Once thesolution reached temperature, surface beta-glucan skins could beharvested about every 15 minutes. More than two dozen skins weresequentially harvested. Samples of three randomly selected skins weretaken and oven-dried. They were found to contain beta-glucan in thefollowing concentrations: 65.5% w, 73.6% w and 65.5% w. After the finalskin was harvested, the depth of the remaining solution was 1.25 inches.This remaining solution contained 28.7% w beta-glucan and would havebeen capable of generating additional beta-glucan surface skins if sodesired. All of the skins were combined and dried on a laboratorydouble-drum dryer (3″ diameter×5″ length). The beta-glucan concentrationwas 67.5% w.

EXAMPLE 3 Drying of Intermediate Solution

[0049] Three successive intermediate solution runs were performedaccording to Example 1. The intermediate solutions from these runs wasevaporated under vacuum until about 75% of the water was lost. Thevessel employed for this purpose was constructed of stainless steel andoperated at 21 inches of vacuum and 99° C. The combined, concentratedsolution was drum dried using a Blaw-Knox double-drum drier withchrome-plated rolls 4 feet in diameter and 10 feet in length. The driedmaterial contained 45.9% w beta-glucan.

EXAMPLE 4 Continuous Flow Ultrafiltration Process

[0050] Commercial oat bran (Con Agra SCM-350) was fed into an Ajax 17Dhammer mill fitted with a 2.5/64-inch screen. The resultant flour wasconveyed through a metered augering system at a rate of 1 lb/min to a20-gallon stainless steel tank where it was slurried with 15° C. waterto approximately 7% w solids concentration. The flour/water slurry waspumped to an oscillatory screen (Rotex Model 3431, 40×84″) fitted withan overhead cold water spray bar and a USS 100 screen. The oversizedmaterial from the screen was gravity-fed to a 230-gallon stainlesssteel, jacketed extractor fitted with a propeller agitator. Sufficientwater was added to maintain 3% w solids concentration. Temperature wasmaintained at 60° C. and pH was maintained continuously at 10 byfeedback-controlled addition of 20% w sodium carbonate solution. The netflow rate was adjusted so that average residence time of the extractionmass was 1 hour. Extraction mass exiting the vessel was pumped to adecanter centrifuge (Bird HP200) in order to remove solids. Supernatantfluid from the centrifuge was discharged to another 230-gallon stainlesssteel jacketed vessel equipped with propeller agitation. Temperature inthis vessel was maintained at 85° C. and pH was continuously maintainedat 7 by feedback-controlled addition of 20% HCl. An amylolytic enzymewas also added (Spezyme Delta AA ex Genencor) at a rate of 10 ml/min.The net flow rate was adjusted so that average residence time of thesupernatant was 30 minutes. Material exiting the vessel was conveyedthrough the shell side of a stainless steel shell-and-tube heatexchanger and was thereby cooled to less than 30° C. The cooled fluidwas pumped to an agitated stainless steel 175-gal. vessel where pH wasmaintained at 4 by feedback-controlled addition of HCl. The fluid wasgravity-fed through a disk centrifuge (Westfalia SAMN 5036) in order toremove precipitated solids. The intermediate solution exiting from thecentrifuge had a characteristic greenish translucent color.

[0051] The intermediate solution was pumped to a 220-gallon stainlesssteel vessel feeding an ultrafiltration unit (U.S. Filter Membraloxceramic filtration unit with P19-40, 0.2:m ceramic elements). Freshwater was added to the vessel at a rate sufficient to offset the lossrate of permeate. The contents of the vessel were maintained at 90° C.through heat exchange with the ultrafiltration unit. Retentate,containing dissolved beta-glucan, was bled off from the ultrafiltrationunit and transferred to a 60-gallon stainless steel surge vessel, thengravity-fed to a double-drum dryer (Drum Dryer & Flaker Corp. Model 28,18×36″ chrome-plated drums). The dried product was subsequently milled,yielding a beige-colored, fine powder. The beta-glucan concentration ofthis final product was determined to be 66.0%. The average molecularweight of the final product was determined to be about 720,000 by sizeexclusion high performance liquid chromatography (HPLC) using a ShodexKB-805 column.

EXAMPLE 5 Formation of Beta-Glucan “Skin” During Continuous FlowUltrafiltration

[0052] The general steps described in the previous example werefollowed. It was noted that a thin, colorless, translucent surface skinreadily formed in the surge vessel feeding the drum dryer. The water inthis vessel was evaporating because of the residual heat contained inthe fluid and the fact that the top of the vessel was uncovered. Becausethe vessel was not equipped with agitation, the surface was quiescent,which may have further facilitated formation of the skin. When a sampleof this skin was analyzed, it was found to contain 87.6% w beta-glucan.When the skin was removed from the surface of the vessel, another layerof skin formed within a few minutes. In contrast, the solids dissolvedin the solution underneath the surface film contained 40.1% wbeta-glucan.

EXAMPLE 6 Effect of Heating Beta-Glucan to Near the Boiling Point

[0053] To determine the effect of the heating step in producing thebeta-glucan intermediate solution, three procedures were performedfollowing the alkaline extraction/centrifugation step (FIG. 1). Allroutes employed the same initial steps: cold slurrying/screening toremove starch fines, followed by alkaline extraction (sodium carbonate,pH 10, 60° C., 1.5 hour) and removal of solids by centrilugation (FIG.1).

[0054] Route 1: Cool

acidify

centrifuge

[0055] This route involves cooling to ambient temperature, followed byacidification with HCl to the isoelectric point (pH 4) and subsequentremoval of precipitated solids by centrifugation. This is theconventional route described in the literature and, typically, precedesbeta-glucan precipitation in alcohol.

[0056] Route 2: Acidify

heat (85° C., 30 min.)

cool

centrifuge

[0057] Route 3: Neutralize (pH 7, 70° C.)

heat (85° C., 30 min.)

cool

acidify

centrifuge

[0058] The results are shown in Table 1. The data in Table 1 representthe average of two identical experiments which produced very similarresults. TABLE 1 Route 1 Route 2 Route 3 Centrifuge pellet weight(dried), g 0.26 2.16 1.88 Beta-glucan in dried supematant, % w 22.7 39.531.5 Total solids in supernatant, % w 1.02 0.78 0.91

[0059] As shown in Table 1, routes 2 and 3, which involve a heatingstep, remove far more contaminants when combined with isoelectricprecipitation than the conventional route (route 1), comprisingisoelectric precipitation only. In fact, the pellet weights resultingfrom routes 2 and 3 are seven to eight times greater. This isunderscored by the significantly higher beta-glucan concentrationsresulting from routes 2 and 3 (74% and 39% higher, respectively). Thetotal dissolved solids found in the supernatants are also consistentwith these findings, with the lowest remnant solids corresponding to thehighest beta-glucan concentration and vice-versa.

[0060] Because of the higher beta-glucan concentration in thesupernatant, route 2 appears preferable to route 3. In addition, thecentrifuge pellet from route 2 was firm, whereas the pellet from route 3was soft. A soft pellet generally indicates more difficult solidsseparation in a setting in which industrial, rather than laboratory,centrifuges are used. Nonetheless, both routes 2 and 3 show considerableutility compared to the conventional route. Although these routes bothemploy a post-extraction heating step, acidification is performed duringheating for route 2 and post-heating for route 3.

EXAMPLE 7 Beta-Glucan Skin Formation Parameter Studies

[0061] Intermediate solution was prepared as described in FIG. 1. Uponanalysis, the total dissolved solids in this material were determined tobe 0.41% w. These solids comprised dissolved beta-glucan (45.1% w), aswell as other dissolved contaminants including protein, starch andinorganic salts. An aliquot of this material (217.6 g) in a shallowglass bowl (11 cm in diameter) was placed in a convection oven operatingat 89° C. The sample was removed from the oven once a surface skin hadformed (within about 30 minutes). It was determined that the totaldissolved solids had increased to only 0.45% w.

[0062] On the other hand, dried beta-glucan concentrate prepared by theprocess of the present invention can be dissolved in water to form ahighly viscous solution containing a maximum of about 1.7% wbeta-glucan. The maximum concentration will most likely depend uponmolecular weight, and the instant process typically produces beta-glucanhaving an average molecular weight of between about 400,000 and onemillion daltons. Presumably, such a solution is at or near thesaturation concentration of beta-glucan. Thus, the beta-glucan skin wasformed from solution at a concentration well below the saturation level.In fact, the beta-glucan concentration in this instance was only about0.2% w, or about 12% of saturation. However, the foregoing does notexclude the possibility of highly localized saturation orsupersaturation conditions.

[0063] The possibility of beta-glucan skin formation occurring via achemical reaction was also considered. Specifically, acidificationassociated with isoelectric precipitation could promote formation of asurface skin by modifying terminal glucose units. To test this,intermediate solution was prepared having an initial pH of 4.3. Aportion of this material was adjusted to approximately neutral pH (7.1)using sodium carbonate. Samples of each material (pH 4.3 and pH 7.1)were placed in an 89° C. convection oven for about one hour, withinwhich time the characteristic beta-glucan skin had formed on the surfaceof the remaining liquid in each container. Accordingly, a pH-drivenchemical reaction does not appear to play a role in surface skinformation.

EXAMPLE 8 Multiple Harvests of Beta-Glucan Skin

[0064] Intermediate solution was prepared substantially as described inExample 1. An aliquot of this solution weighing 1,108 g was placed in aglass bowl, 21 cm in diameter and 9.5 cm high. Initially, the solutioncontained 0.63% solids by weight; the beta-glucan concentration of thesesolids was 49.5% by weight. The bowl was placed on a laboratory hotplate and the solution was brought rapidly to a boil. An initial solidsurface skin formed prior to boiling and was harvested. Whilemaintaining the solution at the boiling point, a series of skins washarvested at intervals of about five minutes over a 3 hour period. Eachsuccessive skin appeared within about two minutes subsequent to eachharvest. After about 1.8 hours, the rate of skin formation slowed as theremaining solution became increasingly briny. Accordingly, 400 ml offresh water was added, resulting in restoration of the previous rapidrate of film regeneration. Surface skins were collected until about 100ml of solution remained; however, additional skins may also be collectedif desired. All of the harvested skins were combined and analyzed. Thebeta-glucan content was determined to be 62.7% by weight, correspondingto an overall beta-glucan recovery of 63.3% from the initial solution.It is contemplated that higher beta-glucan concentration and recoverymay be achieved by water washing the skins and returning the washwaterto the heated vessel. It is also contemplated that a larger evaporationarea will substantially increase the rate of recovery.

[0065] It should be noted that the present invention is not limited toonly those embodiments described in the Detailed Description. Anyembodiment that retains the spirit of the present invention should beconsidered to be within its scope. However, the invention is onlylimited by the scope of the following claims.

What is claimed is:
 1. A physiologically acceptable concentratedbeta-glucan composition comprising mixed (1,3)(1,4) linkages prepared inan alcohol free process in the absence of organic solvents.
 2. Thecomposition of claim 1, wherein the concentration of said beta glucan isgreater than 68%.
 3. The composition of claim 1, wherein saidbeta-glucan is produced by a method comprising: a) providing an alkalineaqueous extract of a beta glucan source; b) acidifying or neutralizingsaid extract and heating said extract to between about 60° C. and 100°C.; c) cooling said extract, whereby a flocculate is formed; d)acidifying said cooled extract if said extract was neutralized in step(b); and e) removing said flocculate from said extract to form abeta-glucan containing solution.
 4. The composition of claim 1, whereinsaid beta glucan is produced by a method comprising heating abeta-glucan containing solution to allow evaporation therefrom, wherebya solid film enriched in beta-glucan is formed on the surface of saidsolution.
 5. The composition of claim 4, wherein said method furthercomprises the step of removing said beta-glucan film, resulting in theformation of a second beta-glucan film.
 6. The composition of claim 5,wherein said beta glucan film removing step is performed one or moretimes.
 7. The composition of claim 4, wherein said method furthercomprises the step of drying said beta-glucan film.
 8. The compositionof claim 1, wherein said beta glucan has a molecular weight of at leastabout 50 kDa.
 9. The composition of claim 1, wherein said beta glucan isselected from those obtainable from oats, barley, wheat, rye, corn,rice, sorghum, millet, or amaranth.
 10. The composition of claim 1,wherein said beta glucan is formulated for oral administration.
 11. Adietary supplement for reducing low density lipoprotein and total serumcholesterol comprising concentrated (1,3)(1,4)-beta glucan prepared inan alcohol free process in the absence of organic solvents.
 12. Thesupplement of claim 11, wherein the concentration of said beta glucan isgreater than 68%.
 13. The supplement of claim 11, wherein saidbeta-glucan is produced by a method comprising: a) providing an alkalineaqueous extract of a beta glucan source; b) acidifying or neutralizingsaid extract and heating said extract to between about 60° C. and 100°C.; c) cooling said extract, whereby a flocculate is formed; d)acidifying said cooled extract if said extract was neutralized in step(b); and e) removing said flocculate from said extract to form abeta-glucan containing solution.
 14. The supplement of claim 11, whereinsaid beta glucan is produced by a method comprising heating a betaglucan containing solution to allow evaporation therefrom, whereby asolid film enriched in beta-glucan is formed on the surface of saidsolution.
 15. The supplement of claim 14, wherein said method furthercomprises the step of removing said beta-glucan film, resulting in theformation of a second beta-glucan film.
 16. The supplement of claim 15,wherein said beta glucan film removing step is performed one or moretimes.
 17. The supplement of claim 14, wherein said method furthercomprises the step of drying said beta-glucan film.
 18. The supplementof claim 17, wherein said film is milled, powdered, dissolved orotherwise dispersed.
 19. The supplement of claim 11, wherein said betaglucan has a molecular weight of at least about 50 kDa.
 20. Thesupplement of claim 1 1, wherein said beta glucan is selected from thoseobtainable from oats, barley, wheat, rye, corn, rice, sorghum, millet,or amaranth.
 21. The supplement of claim 11, wherein said beta glucan isformulated for oral administration.
 22. A composition comprisingconcentrated (1,3)(1,4) beta glucan in a cosmetic composition, whereinsaid beta glucan is prepared in an alcohol free process without the useof organic solvents.
 23. The composition of claim 22, wherein theconcentration of said beta glucan is greater than 68%.
 24. Thecomposition of claim 22, wherein said beta-glucan is produced by amethod comprising: a) providing an alkaline aqueous extract of a betaglucan source; b) acidifying or neutralizing said extract and heatingsaid extract to between about 60° C. and 100° C.; c) cooling saidextract, whereby a flocculate is formed; d) acidifying said cooledextract if said extract was neutralized in step (b); and e) removingsaid flocculate from said extract to form a beta-glucan containingsolution.
 25. The composition of claim 22, wherein said beta glucan isproduced by a method comprising heating a beta glucan containingsolution to allow evaporation therefrom, whereby a solid film enrichedin beta glucan is formed on the surface of said solution.
 26. Thecomposition of claim 25, wherein said method further comprises the stepof removing said beta-glucan film, resulting in the formation of asecond beta-glucan film.
 27. The composition of claim 26, wherein saidbeta glucan film removing step is performed one or more times.
 28. Thecomposition of claim 25, wherein said method further comprises the stepof drying said beta-glucan film.
 29. The composition of claim 28,wherein said film is milled, powdered, dissolved or otherwise dispersedprior to combination with said cosmetic composition.
 30. The compositionof claim 22, wherein said beta glucan has a molecular weight of at leastabout 50 kDa.
 31. The composition of claim 22, wherein said beta glucanis selected from those obtainable from oats, barley, wheat, rye, corn,rice, sorghum, millet, or amaranth.
 32. A composition comprisingconcentrated (1,3)(1,4) beta glucan in combination with a food product,wherein said beta glucan is prepared in an alcohol free process withoutthe use of organic solvents.
 33. The composition of claim 32, whereinthe concentration of said beta glucan is greater than 68%.
 34. Thecomposition of claim 32, wherein said beta glucan is produced by amethod comprising: a) providing an alkaline aqueous extract of a betaglucan source; b) acidifying or neutralizing said extract and heatingsaid extract to betwcen about 60° C. and 100° C.; c) cooling saidextract, whereby a flocculate is formed; d) acidifying said cooledextract if said extract was neutralized in step (b); and e) removingsaid flocculate from said extract to form a beta glucan containingsolution.
 35. The composition of claim 32, wherein said beta glucan isproduced by a method comprising heating a beta-glucan containingsolution to allow evaporation therefrom, whereby a solid film enrichedin beta-glucan is formed on the surface of said solution.
 36. Thecomposition of claim 35, wherein said method further comprises the stepof removing said beta-glucan film, resulting in the formation of asecond beta-glucan film.
 37. The composition of claim 36, wherein saidbeta glucan film removing step is performed one or more times.
 38. Thecomposition of claim 35, wherein said method further comprises the stepof drying said beta-glucan film.
 39. The composition of claim 38,wherein said film is milled, powdered, dissolved or otherwise dispersedprior to combination with said food product.
 40. The composition ofclaim 32, wherein said beta glucan has a molecular weight of at leastabout 50 kDa.
 41. The composition of claim 32, wherein said beta glucanis selected from those obtainable from oats, barley, wheat, rye, corn,rice, sorghum, millet, or amaranth.
 42. A pharmaceutical compositioncomprising concentrated (1,3)(1,4) beta glucan and a pharmaceuticallyacceptable carrier, wherein said beta glucan is prepared in an alcoholfree process without the use of organic solvents.
 43. The composition ofclaim 42, wherein the concentration of said beta glucan is greater than68%.
 44. The composition of claim 42, wherein said beta glucan isproduced by a method comprising: f) providing an alkaline aqueousextract of a beta glucan source; g) acidifying or neutralizing saidextract and heating said extract to between about 60° C. and 100° C.; h)cooling said extract, whereby a flocculate is formed; i) acidifying saidcooled extract if said extract was neutralized in step (b); and j)removing said flocculate from said extract to form a beta glucancontaining solution.
 45. The composition of claim 42, wherein said betaglucan is produced by a method comprising heating a beta-glucancontaining solution to allow evaporation therefrom, whereby a solid filmenriched in beta-glucan is formed on the surface of said solution. 46.The composition of claim 45, wherein said method further comprises thestep of removing said beta-glucan film, resulting in the formation of asecond beta-glucan film.
 47. The composition of claim 46, wherein saidbeta glucan film removing step is performed one or more times.
 48. Thecomposition of claim 45, wherein said method further comprises the stepof drying said beta-glucan film.
 49. The composition of claim 48,wherein said film is milled, powdered, dissolved or otherwise dispersedprior to combination with said food product.
 50. The composition ofclaim 42, wherein said beta glucan has a molecular weight of at leastabout 50 kDa.
 51. The composition of claim 42, wherein said beta glucanis selected from those obtainable from oats, barley, wheat, rye, corn,rice, sorghum, millet, or amaranth.
 52. The composition of claim 42,wherein said beta glucan is formulated for oral administration.